Colorectal cancer (CRC) is the third most prevalent form of cancer in the US and the second leading cause of cancer deaths, with an estimated 50,000 Americans succumbing to CRC in 2015. Studies over the last several decades have revealed that the gut microbiota influences multiple types of cancer, including CRC, and recent work has implicated bacterial genotoxins as key effectors in cancer development and progression. One of the bacterial genotoxins most strongly connected to cancer is colibactin, a metabolite produced by human gut commensal E. coli strains that possess a biosynthetic gene cluster dubbed the pks island. The increased abundance of pks+ E. coli found in CRC and inflammatory bowel disease (IBD) patients and the ability of pks+ strains to potentiate tumorigenesis in mouse models of CRC suggests that colibactin may promote cancer progression. However, achieving a mechanistic understanding of colibactin-mediated DNA damage and genotoxicity has been impeded by an inability to isolate this metabolite or otherwise assign its chemical structure. The overall objective of this proposal is to uncover the molecular mechanism underlying colibactin's genotoxic activity in order to understand and prevent colibactin-mediated carcinogenesis. Preliminary results have revealed that the colibactin biosynthetic pathway assembles structural features found in other DNA- damaging natural products, leading to the hypothesis that colibactin's genotoxicity arises from a direct interaction with DNA. To test this idea and obtain critical knowledge needed to ascertain colibactin's impact on CRC, our three complementary specific aims are to: 1) elucidate the chemical structure of colibactin by integrating multiple isolation strategies; 2) decipher the mechanism by which colibactin exposure leads to DNA damage; and 3) identify small molecules that inhibit colibactin biosynthesis. These advances will be enabled by our multidisciplinary approach, which merges knowledge and techniques from organic chemistry, chemical biology, biosynthesis and enzymology, microbiology, toxicology, and human cell biology. Overall, this effort will generate the tools and knowledge needed to elucidate the role of colibactin-producing bacteria in CRC initiation and progression in humans. By successfully demonstrating that studying and manipulating individual disease-associated microbial metabolic pathways can provide key mechanistic insights, this work will support and validate our future efforts to understand how other gut microbial metabolic activities influence CRC initiation and development.